U.S. patent application number 12/154226 was filed with the patent office on 2009-02-12 for ophthalmic formulations of amyloid-beta contrast agent and methods of use thereof.
Invention is credited to Lee E. Goldstein, Paul D. Hartung, Evan A. Sherr, Francis X. Smith, Kevin R. Sullivan.
Application Number | 20090041666 12/154226 |
Document ID | / |
Family ID | 39671757 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090041666 |
Kind Code |
A1 |
Goldstein; Lee E. ; et
al. |
February 12, 2009 |
Ophthalmic formulations of Amyloid-beta contrast agent and methods
of use thereof
Abstract
The invention provides ophthalmic formulations of Amyloid-.beta.
contrast agents. Also provided are methods of using such
formulations in the diagnosis of Alzheimer's Disease or a
predisposition thereto as well as methods for the prognosis of
Alzheimer's Disease.
Inventors: |
Goldstein; Lee E.; (Newton,
MA) ; Sherr; Evan A.; (Ashland, MA) ; Hartung;
Paul D.; (Acton, MA) ; Smith; Francis X.;
(Salem, NH) ; Sullivan; Kevin R.; (Derry,
NH) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
39671757 |
Appl. No.: |
12/154226 |
Filed: |
May 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60931189 |
May 21, 2007 |
|
|
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61062170 |
Jan 23, 2008 |
|
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Current U.S.
Class: |
424/9.1 ;
514/533; 514/568 |
Current CPC
Class: |
A61K 49/006 20130101;
A61P 27/02 20180101; A61K 31/05 20130101; A61K 31/085 20130101;
A61K 31/194 20130101; A61K 31/216 20130101; A61K 9/0048 20130101;
A61P 25/28 20180101 |
Class at
Publication: |
424/9.1 ;
514/568; 514/533 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 31/194 20060101 A61K031/194; A61P 27/02 20060101
A61P027/02; A61K 31/216 20060101 A61K031/216 |
Claims
1. An ophthalmic formulation comprising an effective amount of a
compound of Formula I and a pharmaceutically acceptable carrier or
excipient, wherein the formulation has an octanol-water partition
coefficient K.sub.ow of between 100 and 300 or a LogD value of
between 1 and 3 ##STR00016## wherein R'.sub.2 is selected from the
group consisting of H, OH, and OCH.sub.3; is selected from the
group consisting of H, COOH, and CO.sub.2CH.sub.3; and R.sub.4 is
selected from the group consisting of H, OH, and OCH.sub.3.
2. The formulation of claim 1, wherein the compound of Formula I is
selected from the group consisting of compounds of Formula II,
Formula III, Formula VIII, and Formula X. ##STR00017##
3. The formulation of claim 1, wherein the formulation is soluble
in the cornea, aqueous humor, and lens of the eye.
4. The formulation of claim 1, wherein the formulation further
comprises a preservative.
5. The formulation of claim 4, wherein the preservative is present
in a concentration of less than 1%.
6. The formulation of claim 1, wherein the formulation further
comprises a pupil dilating agent.
7. The formulation of claim 6, wherein the pupil dilating agent is
a mydriatic.
8. The formulation of claim 7, wherein the mydriatic is
atropine.
9. The formulation of claim 1, wherein the K.sub.ow is between 200
and 300.
10. The formulation of claim 2, wherein the compound of Formula X
comprises particles less than 6 .mu.m in size.
11. An ophthalmic formulation, wherein said formulation is an
ointment comprising an effective amount of a compound of Formula I
and a pharmaceutically acceptable carrier or excipient, wherein
said formulation has a logP.sub.oct value less than 2.6
##STR00018## wherein R'.sub.2 is selected from the group consisting
of H, OH, and OCH.sub.3; R.sub.3 is selected from the group
consisting of H, COOH, and CO.sub.2CH.sub.3; and R.sub.4 is
selected from the group consisting of H, OH, and OCH.sub.3.
12. The formulation of claim 11, wherein said formulation is
soluble in the cornea, aqueous humor, and lens of the eye.
13. The formulation of claim 11, wherein the compound of Formula I
is selected from the group consisting of the compound of Formula
VIII and the compound of Formula X. ##STR00019##
14. The formulation of claim 13, wherein the compound of Formula I
is the compound of Formula X.
15. The formulation of claim 11, wherein the excipient is selected
from the group consisting of petrolatum, mineral oil, or
combinations thereof.
16. The formulation of claim 15, wherein the formulation comprises
1% or less of the hydrophobic compound of Formula I, 85%
petrolatum, and 15% mineral oil.
17. The formulation of claim 11, wherein the formulation further
comprises a preservative.
18. The formulation of claim 17, wherein the preservative is
present in a concentration of less than 1%.
19. The formulation of claim 11, wherein the formulation further
comprises a pupil dilating agent.
20. The formulation of claim 19, wherein the pupil dilating agent
is a mydriatic.
21. The formulation of claim 20, wherein the mydriatic is
atropine.
22. The formulation of claim 11, wherein the excipient is an
aqueous solution comprising a viscosity agent.
23. The formulation of claim 22, wherein the formulation comprises:
(a) 1% or less of the compound of Formula I; (b) a surfactant
comprising polysorbate 80; (c) a preservative comprising
benzalkonium chloride; (d) a tonicity agent comprising sodium
chloride; (e) a buffer comprising boric acid or a salt thereof; (f)
a chelating agent comprising edentate disodium; and (g) a viscosity
agent comprising hydroxypropyl methylcellulose.
24. The formulation of claim 22, wherein the formulation comprises
a preservative.
25. The formulation of claim 24, wherein the preservative is
present in a concentration of less than 1%.
26. The formulation of claim 22, wherein the formulation further
comprises a pupil dilating agent.
27. The formulation of claim 26, wherein the pupil dilating agent
is a mydriatic.
28. The formulation of claim 27, wherein the mydriatic is
atropine.
29. The formulation of claim 14, wherein the compound of Formula X
comprises particles less than 6 .mu.m in size.
30. An ophthalmic formulation, wherein said formulation is an
aqueous solution comprising an effective amount of a compound of
Formula I and a pharmaceutically acceptable carrier or excipient,
wherein said formulation has a LogD value less than 0.42
##STR00020## wherein R'.sub.2 is selected from the group consisting
of H, OH, and OCH.sub.3; R.sub.3 is selected from the group
consisting of H, COOH, and CO.sub.2CH.sub.3; and R.sub.4 is
selected from the group consisting of H, OH, and OCH.sub.3.
31. The formulation of claim 30, wherein said formulation is
soluble in the cornea, aqueous humor, and lens of the eye.
32. The formulation of claim 30, wherein the compound of Formula I
is selected from the group consisting of the compound of Formula II
and the compound of Formula III. ##STR00021##
33. The formulation of claim 32, wherein the compound of Formula I
is the compound of Formula II.
34. The formulation of claim 30, wherein the formulation comprises
a buffered aqueous excipient.
35. The formulation of claim 34, wherein the buffered aqueous
excipient comprises water, propylene glycol, or both.
36. The formulation of claim 35, wherein the formulation comprises
a buffer to provide proper pH for maximum solubility of said
compound of Formula I, a chelating agent, and a preservative.
37. The formulation of claim 36, wherein said buffer is Tris,
wherein said chelating agent is ethylenediamine-tetraacetate, and
wherein said preservative is parabens.
38. The formulation of claim 36, wherein the formulation comprises
a) 1% or less of the compound of Formula I; b) a solvent comprising
water; c) 0.001% to 10% Tris-buffer; d) 0.001% to 1% EDTA; and e)
0.0001% to 1% parabens.
39. The formulation of claim 36, wherein the preservative is
selected from the group consisting of propyl paraben and
benzalkonium chloride.
40. The formulation of claim 36, wherein the preservative is
present in a concentration of less than 1%.
41. The formulation of claim 30, wherein the formulation further
comprises a pupil dilating agent.
42. The formulation of claim 41, wherein the pupil dilating agent
is a mydriatic.
43. The formulation of claim 42, wherein the mydriatic is
atropine.
44. The formulation of claim 30, wherein the formulation further
comprises a thickening agent.
45. The formulation of claim 44, wherein the thickening agent is
selected from the group consisting of cellulose derivative
thickening agents, hydroxypropyl methylcellulose, methylcellulose,
hydroxyethyl cellulose, non-cellulose thickening agents, polyvinyl
pyrrolidone, polyacrylates, and carbomes.
46. The formulation of claim 44, wherein the thickening agent
increases the viscosity of the formulation up to 1,000,000
centiPoise.
47. The formulation of claim 46, wherein the thickening agent
increases the viscosity of the formulation to between 10 and 1000
centipoise.
48. An ophthalmic formulation comprising less than about 2% of a
compound of Formula I and a pharmaceutically acceptable carrier.
##STR00022## wherein R'.sub.2 is selected from the group consisting
of H, OH, and OCH.sub.3; R.sub.3 is selected from the group
consisting of H, COOH, and CO.sub.2CH.sub.3; and R.sub.4 is
selected from the group consisting of H, OH, and OCH.sub.3.
49. The formulation of claim 48, wherein the formulation comprises
less than about 0.1% of the compound of Formula I.
50. The formulation of claim 48, wherein the compound of Formula I
is selected from the group consisting of the compounds of Formula
II, Formula III, Formula VIII, and Formula X. ##STR00023##
51. The formulation of claim 48, wherein the formulation has an
octanol-water partition coefficient K.sub.ow of between 100 and 300
or a LogD value of between 1 and 3.
52. The formulation of claim 51, wherein the formulation is soluble
in the cornea, aqueous humor, and lens of the eye.
53. The formulation of claim 48, wherein the formulation is in the
form of a tape, an ointment, an eye drop, or an aqueous
solution.
54. The formulation of claim 48, wherein the formulation further
comprises a preservative selected from the group consisting of
propyl paraben and benzalkonium chloride.
55. The formulation of claim 54, wherein the preservative is
present in a concentration of less than 1%.
56. The formulation of claim 48, wherein the formulation further
comprises a pupil dilating agent.
57. The formulation of claim 56, wherein the pupil dilating agent
is a mydriatic.
58. The formulation of claim 57, wherein the mydriatic is
atropine.
59. The formulation of claim 50, wherein the compound of Formula X
comprises particles less than 6 .mu.m in size.
60. A method of diagnosing Alzheimer's Disease or a predisposition
thereto in a mammal, comprising (a) contacting an ocular tissue
with the ophthalmic formulation of any one of claims 1, 11, 30, or
48; (b) allowing said formulation to distribute into the lens; and
(c) imaging said ocular tissue, wherein an increase in binding of
said formulation to said ocular tissue compared to a normal control
level of binding indicates that said mammal is suffering from or is
at risk of developing Alzheimer's Disease.
61. The method of claim 60, wherein said ocular tissue comprises a
cortical region of an eye.
62. The method of claim 60, wherein said ocular tissue comprises a
supranuclear region of an eye.
63. The method of claim 60, wherein said ocular tissue comprises an
aqueous humor region of an eye.
64. The method of claim 60, wherein said increase is at least 10%
greater than said normal control value.
65. The method of claim 60, wherein said increase is at least 25%
greater than said normal control value.
66. The method of claim 60, wherein said increase is at least 50%
greater than said normal control value.
67. The method of claim 60, wherein said increase is at least 100%
greater than said normal control value.
68. The method of claim 60, wherein the formulation is applied to
the cornea and is able to diffuse through the cornea and the
aqueous humor to the lens of the eye.
69. The method of claim 60, wherein said contacting in step (a)
occurs via topical administration of said ophthalmic
formulation.
70. The method of claim 60, wherein said contacting in step (a)
occurs via injection of said ophthalmic formulation.
71. A method for prognosis of Alzheimer's Disease, comprising (a)
contacting ocular tissue of a mammal with the ophthalmic
formulation of any one of claims 1, 11, 30, or 48; (b) allowing
said formulation to distribute into the lens (c) imaging said
ocular tissue; (d) quantitating the level of association of said
formulation with said ocular tissue; and (e) comparing said level
of association with a normal control level of association, wherein
increasing levels of association over time indicates an adverse
prognosis.
72. A method of diagnosing Alzheimer's Disease or a predisposition
thereto in a mammal, comprising (a) administering the formulation
of any one of claims 10, 29, or 59 to the mammal; (b) allowing said
formulation to distribute into the lens of the eye; and (c) imaging
an ocular tissue, wherein an increase in binding of said
formulation to said ocular tissue compared to a normal control
level of binding indicates that said mammal is suffering from or is
at risk of developing Alzheimer's Disease.
73. The method of claim 72, wherein said ocular tissue comprises a
cortical region, a supranuclear region, or an aqueous humor region
of an eye.
74. The method of claim 72, wherein said increase is at least 10%
greater than said normal control value.
75. The method of claim 72, wherein said increase is at least 25%
greater than said normal control value.
76. The method of claim 72, wherein said increase is at least 50%
greater than said normal control value.
77. The method of claim 72, wherein said increase is at least 100%
greater than said normal control value.
78. The method of claim 72, wherein the formulation is administered
systemically.
79. The method of claim 78, wherein the formulation is administered
via systemic injection.
80. The method of claim 72, wherein the formulation is administered
ocularly.
81. The method of claim 80, wherein the formulation is administered
via ocular injection.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Ser. No.
60/931,189, filed May 21, 2007 and U.S. Ser. No. 61/062,170, filed
Jan. 23, 2008. Each of these applications is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to neurodegenerative
disease.
BACKGROUND
[0003] Alzheimer's Disease ("AD") is a chronically progressive
degenerative disorder of aging and is a major contributor to
morbidity and modality in the elderly. AD currently accounts for
about 70% of all cases of dementia and affects some 2-4 million
Americans. As many as 9 million Americans may have AD by the year
2050. Epidemiological studies have estimated that if the onset of
AD could be delayed by 5 years, the incidence and prevalence of AD
would be cut in half.
[0004] The accumulation of Amyloid-.beta. ("A.beta.") has been
implicated in the pathogenesis of Alzheimer's disease. A.beta. has
also been shown to accumulate in the lens of the eye at levels,
which make the detection of A.beta. aggregations in the lens a
useful method of diagnosing and evaluating Alzheimer's Disease
progress.
SUMMARY OF THE INVENTION
[0005] Congo Red- and Chrysamine G-derivatives such Methoxy-X04 and
X34 have previously been used as in vivo contrast agents to detect
amyloid-.beta. plaques in the brain. These compounds are not
suitable for administration to the eye because of their
bioavailabilty, solubility, and/or toxicity characteristics.
Accordingly, in order to successfully use these derivative
compounds as in vivo contrast agents for detection of A.beta.
accumulation in the lens of the eye, the compounds are incorporated
into ophthalmic formulations having improved bioavailabilty
characteristics, while still retaining the A.beta.-binding
characteristics of the derivatives.
[0006] For example, ophthalmic formulations contain an effective
amount of a compound of Formula I along with a pharmaceutically
acceptable carrier or excipient. Those skilled in the art will
recognize that, in Formula I, R'.sub.2 can be selected from the
group consisting of H, OH, and OCH.sub.3; R.sub.3 can be selected
from the group consisting of H, COOH, and CO.sub.2CH.sub.3; and
R.sub.4 can be selected from the group consisting of H, OH, and
OCH.sub.3. These ophthalmic formulations are designed to be applied
to the cornea and diffuse through the cornea and the aqueous humor
to the lens of the eye. Moreover, these ophthalmic formulations are
soluble in the cornea, aqueous humor, and lens of the eye. Such
formulations preferably have an octanol-water partition coefficient
K.sub.ow of between 100 and 300 or a LogD value of between 1 and 3.
For example, the K.sub.ow is 100, 125, 150, 175, 200, 225, 250, 275
or 300 or LogD value is 1, 1.25, 1.50, 1.75, 2, 2.25, 2.5, 2.75, or
3. Preferably, the K.sub.ow is between 200 and 300. Suitable
compounds include, but are not limited to, the compounds of Formula
II, Formula III, Formula VIII, and/or Formula X.
[0007] The formulation may also contain a preservative, such as,
for example, propyl paraben or benzalkonium chloride, which, when
present, is optimally added in a concentration of less than 1%.
Moreover, the ophthalmic formulation may also contain a pupil
dilating agent (i.e., a mydriatic, such as atropine) in order to
provide an optimal field of view for the associated eye test. When
the formulation includes the compound of Formula X, the compound of
Formula X contains particles less than 6 .mu.m in size (i.e., less
than 5 .mu.m, less than 4 .mu.m, less than 3 .mu.m, less than 2
.mu.m, less than 1 .mu.m).
[0008] Some Congo Red- and/or Chrysamine G-derivatives are more
hydrophobic (and, thus, less water soluble) than others. Therefore,
the design of ophthalmic formulations containing this type of
derivative compound must take this relative hydrophobicity into
account. Ophthalmic formulations containing this class of compounds
may be in the form of an ointment containing an effective amount of
a compound of Formula I along with a pharmaceutically acceptable
carrier or excipient. Those skilled in the art will recognize that,
in Formula I, R'.sub.2 can be selected from the group consisting of
H, OH, and OCH.sub.3; R.sub.3 can be selected from the group
consisting of H, COOH, and CO.sub.2CH.sub.3; and R.sub.4 can be
selected from the group consisting of H, OH, and OCH.sub.3. Such
ointments preferably have a logP.sub.oct value less than 2.6 and
are applied to the cornea and diffuse through the cornea and
aqueous humor to the lens of the eye. Moreover, these formulations
are soluble in the cornea, aqueous humor, and lens of the eye.
Exemplary compounds of Formula I for use in such ointments include
the compound of Formula VIII (e.g., X04) and the compound of
Formula X (e.g., Methoxy-X04). Preferably, the hydrophobic compound
of Formula I is the compound of Formula X. When the formulation
includes the compound of Formula X, the compound of Formula X
contains particles less than 6 .mu.m in size (i.e., less than 5
.mu.m, less than 4 .mu.m, less than 3 .mu.m, less than 2 .mu.m,
less than 1 .mu.m).
[0009] The excipient used in the preparation of the ointment
includes petrolatum, mineral oil, or combinations thereof. For
example, one such suitable ophthalmic formulation contains 1% or
less of the hydrophobic compound of Formula I, 85% petrolatum, and
15% mineral oil.
[0010] Optionally, the ointment may also contain a preservative,
such as, for example, propyl paraben or benzalkonium chloride. When
present, the preservative is included in a concentration of less
than 1%. Likewise, the ointment may further optionally contain a
pupil dilating agent, for example a mydriatic such as atropine.
[0011] Alternatively, the excipient used with these relatively
hydrophobic compounds is an aqueous solution comprising a viscosity
agent or an emulsifier. For example, the viscosity agent is
hydroxypropyl methyl cellulose. Such formulations contain less than
about 1% of a preservative selected from the group consisting of
propyl paraben or benzalkonium chloride; and may also optionally
contain a pupil dilating agent (e.g., a mydriatic such as, for
example, atropine).
[0012] For example, one such aqueous solution formulation contains
1% or less of the compound of Formula I; surfactant such as
polysorbate 80; a preservative such as benzalkonium chloride; a
tonicity agent such as sodium chloride; a buffer such as boric acid
or a salt thereof; a chelating agent such as edentate disodium; and
a viscosity agent such as hydroxypropyl methylcellulose. The pH is
adjusted with acids or bases, such as hydrochloric acid or sodium
hydroxide such that the tonicity of the formulation is isotonic
relative to the tissue of the eye, thereby causing little or no
swelling or contraction of the target tissue. Alternative tonicity
agents include boric acid, sodium bicarbonate, and sodium chloride.
Moreover, use of an isotonic formulation also results in little or
no discomfort upon contact of the eye.
[0013] Some Congo Red- and Chrysamine G-derivatives such as X34 are
relatively more hydrophilic in nature compared to either the parent
compounds from which they are derivated or to the other CR- and
CG-derivatives. Ophthalmic formulations containing such derivative
compounds are preferably in the form of aqueous solutions
containing an effective amount of a compound of Formula I and a
pharmaceutically acceptable carrier or excipient and have a LogD
value less than 0.42. Those skilled in the art will recognize that,
in Formula I, R'.sub.2 can be selected from the group consisting of
H, OH, and OCH.sub.3; R.sub.3 can be selected from the group
consisting of H, COOH, and CO.sub.2CH.sub.3; and R.sub.4 can be
selected from the group consisting of H, OH, and OCH.sub.3. These
formulations are designed to be applied to the cornea and are able
to diffuse through the cornea and aqueous humor to the lens of the
eye. Moreover, these formulations are soluble in the cornea,
aqueous humor, and lens of the eye. Suitable compounds for use in
such ophthalmic formulations include, for example, the compound of
Formula II (e.g. X34) and the compound of Formula III (e.g.
Methoxy-X34). Preferably, the compound of Formula I is the compound
of Formula II.
[0014] Such aqueous solution ophthalmic formulations may contain a
preservative (e.g., less than about 1% of propyl paraben or
benzalkonium chloride). In addition, these formulations may also
contain a pupil dilating agent. For example, the pupil dilating
agent may be a mydriatic, such as, for example, atropine.
[0015] In some embodiments, these aqueous solution formulations
contain a buffered aqueous excipient. By way of non-limiting
example, the buffered aqueous excipient is water, propylene glycol,
or both. The presence of the buffer provides proper pH for maximum
solubility of the compound of Formula I, and the formulation may
also contain a chelating agent to improve stability as well as a
preservative. Specifically, the buffer is, for example, Tris, the
chelating agent is, for example, ethylenediamine-tetraacetate, and
the preservative is, for example, parabens. For example, one
preferred aqueous solution formulation described herein may contain
1% or less of the compound of Formula I; a solvent such as water;
0.001% to 10% (e.g., 0.001%, 0.005%, 0.010%, 0.05%, 0.1%, 0.5%, 1%,
2.5%, 5%, 7.5%, or 10%) Tris-buffer; 0.001% to 1% (e.g., 0.001%,
0.005%, 0.010%, 0.025%, 0.05%, 0.1%, 0.5%, 0.75%, or 1%) EDTA; and
0.0001% to 1% (e.g., 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%,
0.05%, 0.1%, 0.11%, 0.5%, or 1%) parabens.
[0016] These aqueous solution formulations optionally contain a
thickening agent to improve the ease of administration, to improve
drug residence time in the eye, or both. By way of non-limiting
example, the thickening agent can a cellulose derivative thickening
agent such as hydroxypropyl methylcellulose, methylcellulose, or
hydroxyethyl cellulose or a non-cellulose thickening agents such as
polyvinyl pyrrolidone, polyacrylates, or carbomes. Those skilled in
the art will recognize that the thickening agent can be used to
increase the viscosity of the formulation up to 1,000,000
centiPoise. Preferably, the viscosity is increased up to 10
centipoise, up to 20 centiPoise, up to 30 centipoise, up to 40
centiPoise, up to 50 centipoise, up to 100 centiPoise, up to 250
centiPoise, up to 500 centipoise, up to 750 centiPoise, or up to
1000 centiPoise. In one preferred embodiment, the viscosity is
increased to between 10 and 1000 centiPoise (i.e., to 10, 20, 25,
30, 40, 50, 75, 100, 250, 500, 750, or 1000 centiPoise).
[0017] Ophthalmic formulations containing the Congo Red- and
Chrysamine G-derivatives may contain less than about 2%, less than
about 1.5%, less than about 1%, or less than about 0.5% of a
compound of Formula I along with a pharmaceutically acceptable
carrier. Those skilled in the art will recognize that, in Formula
I, R'.sub.2 can be selected from the group consisting of H, OH, and
OCH.sub.3; R.sub.3 can be selected from the group consisting of H,
COOH, and CO.sub.2CH.sub.3; and R.sub.4 can be selected from the
group consisting of H, OH, and OCH.sub.3. For example, such a
formulation will contain less than about 0.1% of the compound of
Formula I. Suitable compounds of Formula I that are used in such
ophthalmic formulations include, for example, the compounds of
Formula II, Formula III, Formula VIII, and/or Formula X. When the
formulation includes the compound of Formula X, the compound of
Formula X contains particles less than 6 .mu.m in size (i.e., less
than 5 .mu.m, less than 4 .mu.m, less than 3 .mu.m, less than 2
.mu.m, less than 1 .mu.m).
[0018] For optimal bioavailability for use as an in vivo ocular
contrast agent, these formulations have an octanol-water partition
coefficient K.sub.ow of between 100 and 300 or a LogD value of
between 1 and 3 and are designed to be applied to the cornea and
diffuse through the cornea and the aqueous humor to the lens of the
eye. For example, the K.sub.ow is 100, 125, 150, 175, 200, 225,
250, 275 or 300 or LogD value is 1, 1.25, 1.50, 1.75, 2, 2.25, 2.5,
2.75, or 3. Moreover, these formulations are soluble in the cornea,
aqueous humor, and lens of the eye. Preferably, the formulations
are in the form of a tape, an ointment, an eye drop, or an aqueous
solution.
[0019] These ophthalmic formulations can also contain a
preservative, such as, for example, propyl paraben or benzalkonium
chloride. When added to the ophthalmic formulation, the
preservative is typically present in a concentration of less than
1%. The inclusion of a pupil dilating agent (e.g., a mydriatic such
as atropine) can provide an optimal field of view for the
associated eye test.
[0020] Also provided herein are methods of diagnosing Alzheimer's
Disease or a predisposition thereto in a mammal using any of the
ophthalmic formulations disclosed herein. Specifically, an ocular
tissue (e.g., a deep cortical region, a supranuclear region, or an
aqueous humor region of an eye) is contacted with the ophthalmic
formulation, which is allowed to distribute into the lens. Those
skilled in the art will recognize that any suitable method(s) of
administration or application of the ophthalmic formulations of the
invention (e.g., topical, injection, parenteral, airborne, oral,
and/or suppository administration, etc.) can be employed. For
example, the contacting may occur via topical administration or via
injection. Ocular tissue is then imaged using any imaging technique
known to those in the art. An increase in binding of the ophthalmic
formulation to the ocular tissue compared to a normal control level
of binding indicates that the mammal is suffering from or is at
risk of developing Alzheimer's Disease.
[0021] For example, the increase may be at least 10% greater, at
least 25% greater, at least 50% greater, at least 100%, 3-fold,
5-fold, 10-fold, or more greater than said normal control
value.
[0022] The invention also encompasses a method for prognosis of
Alzheimer's Disease by contacting an ocular tissue of a mammal with
any of the ophthalmic formulations described herein; allowing the
formulation to distribute into the lens; imaging the ocular tissue;
quantitating the level of association of the formulation with the
ocular tissue; and comparing this level of association with a
normal control level of association. Increasing levels of
association over time indicates an adverse Alzheimer's Disease
prognosis. Those skilled in the art will recognize that any
suitable method(s) of administration or application of the
ophthalmic formulations of the invention (e.g., topical, injection,
parenteral, airborne, oral, and/or suppository administration,
etc.) can be employed. For example, the contacting may occur via
topical administration or via injection.
[0023] The methods are also useful to monitor the effect of a
therapeutic intervention; a decrease in the level of association
indicates that the intervention is efficacious, i.e., an
improvement in disease status, whereas an increase indicates a
worsening of the disease or that the intervention is not leading to
a measurable clinical benefit.
[0024] Likewise, the invention also encompasses methods of
diagnosing Alzheimer's Disease or a predisposition thereto in a
mammal by administering any of the formulations of the invention
containing the compound of Formula X, wherein the particles of the
compound of Formula X are less than 6 .mu.m in size of any one of
claims to the mammal; allowing the formulation to distribute into
the lens of the eye; and imaging an ocular tissue (e.g., a cortical
region, a supranuclear region, or an aqueous humor region of an
eye), wherein an increase in binding of the formulation to the
ocular tissue compared to a normal control level of binding
indicates that the mammal is suffering from or is at risk of
developing Alzheimer's Disease.
[0025] For example, the increase may be at least 10% greater, at
least 25% greater, at least 50% greater, or at least 100% greater
than said normal control value. Those skilled in the art will
recognize that such a formulation can be administered systemically
(i.e., via system injection) or ocularly (i.e., via ocular
injection).
[0026] The invention also provides methods of determining the level
of binding of any of the ophthalmic formulations of the invention
to ocular tissue, by imaging the ocular tissue after the ophthalmic
formulation has been administered and allowed to distribute into
the lens of the eye, and comparing the level of binding of said
formulation to the ocular tissue to a normal control level of
binding.
[0027] Finally, also provided is a method of generating a
diagnostic index for predicting the development and/or progression
of a disease or disorder (e.g., Alzheimer's Disease). For example,
the diagnostic index may be generated by collecting a
representative number of values or data points such that a
determination as to illness or wellness can be made for a given
patient. The invention also encompasses the resultant diagnostic
index (e.g., the plurality or collection of values that is
obtained).
[0028] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only not intended to be limiting. Other
features and advantages of the invention will be apparent from the
following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0029] This invention provides for sensitive means to
non-invasively, safely, and reliably detect a biomarker of
Alzheimer's Disease in the lens and other ocular tissues using a
quasi-elastic light scattering, Raman spectroscopy, fluorometric or
any other suitable optical technologies. These techniques allow
detection and monitoring of amyloid protein deposition in the eye
for the diagnosis of neurodegenerative disorders such as AD and
prionopathies. Lens protein aggregation is potentiated by human
A.beta..sub.1-42 peptide, a pathogenic and neurotoxic peptide
species which aggregates and accumulates in AD brain. A.beta. was
found to promote protein aggregation both in vivo and in vitro, and
A.beta..sub.1-42 was found specifically in the deep cortex and
supranucleus of human lenses and was associated with large
molecular weight protein aggregates. The results indicate that the
protein aggregation in the lens, e.g., in lens cortical fiber
cells, represents an easily accessible peripheral marker of AD
pathology in the brain. (See, U.S. Pat. No. 7,107,092, which is
herein incorporated by reference in its entirety).
[0030] Methods of diagnosing, prognosing, staging, and/or
monitoring mammalian amyloidogenic disorders or predisposition
thereto are carried out by detecting a protein or polypeptide
aggregate in the cortical and/or supranuclear region of an ocular
lens of the mammal. This determination is compared to or normalized
against the same determinations in the nuclear region of the same
lens where more general effects of aging are observed. Comparisons
are also made to a population norm, e.g., data compiled from a pool
of subjects with and without disease. The presence of or an
increase in the amount of aggregate in the supranuclear and/or
cortical lens regions of the test mammal compared to a normal
control value indicates that the test mammal is suffering from, or
is at risk of, developing an amyloidogenic disorder. A normal
control value corresponds to a value derived from testing an
age-matched individual known to not have an amyloidogenic disorder
or a value derived from a pool of normal, healthy (e.g. non-AD)
individuals.
[0031] As used herein an "amyloidogenic disorder" is one that is
characterized by deposition or accumulation of an amyloid protein
or fragment thereof in the brain of an individual. Amyloidogenic
disorders include, for example, AD, Familial AD, Sporadic AD,
Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease,
spongiform encephalopathies, Prion diseases (including scrapie,
bovine spongiform encephalopathy, and other veterinary
prionopathies), Parkinson's disease, Huntington's disease (and
trinucleotide repeat diseases), amyotrophic lateral sclerosis,
Downs Syndrome (Trisomy 21), Pick's Disease (Frontotemporal
Dementia), Lewy Body Disease, neurodegeneration with brain iron
accumulation (Hallervorden-Spatz Disease), synucleinopathies
(including Parkinson's disease, multiple system atrophy, dementia
with Lewy Bodies, and others), neuronal intranuclear inclusion
disease, tauopathies (including progressive supranuclear palsy,
corticobasal degeneration, hereditary frontotemporal dementia (with
or without Parkinsonism), and Guam amyotrophic lateral
sclerosis/parkinsonism dementia complex). These disorders may occur
alone or in various combinations. For example, individuals with AD
are characterized by extensive accumulation of amyloid in the brain
in the form of senile plaques, which contain a core of amyloid
fibrils surrounded by dystrophic neurites. Some of these patients
also exhibit clinical signs and symptoms, as well as
neuropathological hallmarks, of Lewy Body disease.
[0032] The presence of and/or an increase in the amount of an
amyloid protein or polypeptide detected in a subject's eye tissue
over time indicates a poor prognosis for disease, whereas absence
or a decrease over time indicates a more favorable prognosis. For
example, a decrease in the amount or a decrease in the rate of
accumulation in amyloid protein or similar changes in the
associated ocular morphological features in eye tissue after
therapeutic intervention indicates that the therapy has clinical
benefit. Therapeutic intervention includes drug therapy such as,
for example, administration of a secretase inhibitor, vaccine,
antioxidant, anti-inflammatory, metal chelator, or hormone
replacement or non-drug therapies.
[0033] Mammals to be tested include human patients, companion
animals such as dogs and cats, and livestock such as cows, sheep,
pigs, horses and others. For example, the methods are useful to
non-invasively detect bovine spongiform encephalopathy (mad cow
disease), scrapie (sheep), and other prionopathies of veterinary
interests.
[0034] For example, the diagnostic test is administered to a human
who has a positive family history of familial AD or other risks
factors for AD (such as advanced age) or is suspected of suffering
from an amyloidogenic disorder, e.g., by exhibiting impaired
cognitive function, or is at risk of developing such a disorder.
Subjects at risk of developing such a disorder include elderly
patients, those who exhibit dementia or other disorders of thought
or intellect, as well as patients with a genetic risk factor.
[0035] A disease state is indicated by the presence of amyloid
protein aggregates or deposits in the supranuclear or deep cortical
regions of a mammalian lens. For example, the amount of amyloid
protein aggregates is increased in a disease state compared to a
normal control amount, i.e., an amount associated with a
nondiseased individual. Amyloid proteins include, for example,
.beta. amyloid precursor protein (APP), A.beta., or a fragment
thereof (e.g., A.beta..sub.1-42), as well as prion proteins, and
synuclein. Protein or polypeptide aggregates may contain other
proteins in addition to A.beta. (such as .alpha., .beta., and or
.gamma. crystallin). Unlike amyloid protein deposition in brain
tissue, which is primarily extracellular, ocular deposition in lens
cortical fiber cells is cytosolic.
Amyloid Imaging Agents
[0036] Congo Red ("CR") is an amyloid-staining agent that is widely
used in post-mortem histological identification, evaluation, and/or
diagnosis of Alzheimer's Disease. (See Mathis et al., Current
Pharmaceutical Design 10(13):1469-92 (2004), incorporated herein by
reference in its entirety). CR selectively binds to A.beta.
aggregations with high affinity. Unfortunately, CR does not have
adequate bioavailability characteristics, which makes it unsuitable
for use as an in vivo contrast agent.
[0037] Chrysamine G ("CG") is a carboxylic acid analogue of CR that
was developed to address (and overcome) some of these shortcomings
of CR. Chrysamine G is considerably more lipophilic than Congo Red.
Thus, it can cross the blood-brain barrier, and it is useful as a
probe for detecting senile plaques (A.beta. aggregates) in the
brain.
[0038] Those skilled in the art will recognize that the
hydrophobicity and hydrophilicity of a substance can be measured
using the octanol-water partition coefficient ("P.sub.oct" or
"K.sub.ow"), for compounds whose solubility is not altered by the
pH and ionic characteristic of the solute, or LogD value, for
compounds whose solubility is altered by the pH and ionic
characteristic of the solute. LogD is the logarithm of the
distribution coefficient, which is the ratio of the sum of the
concentrations of all species of a compound in octanol to the sum
of the concentrations of all species of the compound in water.
[0039] In these contexts, hydrophobicity is related to factors such
as absorption, bioavailabity, hydrophobic drug-receptor
interactions, metabolism, and/or toxicity. As used herein, the
logP.sub.oct is synonymous to logK.sub.ow, and both measurements
are used interchangeably herein. Likewise, logP.sub.oct is a
functionally equivalent measure to LogD; both values reflect the
degree of solubility of a given compound.
[0040] The octanol-water partition coefficient of CR at pH 7.4 is
only 0.7 (logP.sub.oct=0.18), whereas the P.sub.oct of CG is nearly
100-fold higher (P.sub.oct=60; logP.sub.oct=1.8).
[0041] In an effort to develop improved in vivo contrast agents, as
early as 1998, Klunk et. al described a class of Congo Red or
Chrysamine G derivatives that retain their A.beta.-binding
characteristics while improving systemic bioavailability. (See
Klunk et al., Life Sci. 63(20):1807-14 (1998)) The resulting
Chrysamine G derivatives are described in U.S. Pat. Nos. 6,133,259;
6,168,776; and 6,114,175, which are herein incorporated by
reference in their entireties. See also Mathis et al., Current
Pharmaceutical Design 10(13):1469-92 (2004), incorporated herein by
reference. These Chrysamine G derivatives exhibit low systemic
toxicity as well as modest to poor aqueous solubility (e.g.,
logP.sub.oct=1.8) and modest lipid solubility (e.g.,
logP.sub.oct=1.8).
[0042] Examples of such CG derivatives are shown in Table 1. Table
1 also contains a general structure (Formula I) that describes the
exemplary CG derivatives. (See Mathos, et al., Current
Pharmaceutical Design 10: 1469-92 (2004), herein incorporated by
reference).
TABLE-US-00001 TABLE 1 (I) ##STR00001## 2'-Position 3-Position
Compound (R.sub.2') (R.sub.3) 4-Position (R.sub.4) Ki (nM) logPoct
MW Structure X:E:B34 (X-34) H COOH OH 18 0.42 402 ##STR00002##
X:E:B34 bis(4-methoxy) H CO.sub.2CH.sub.3 OCH.sub.3 Inact 1.2 458
##STR00003## dimethyl ester Acid Series X:E:B34 bis(4-methoxy) H
COOH OCH.sub.3 47 -0.95 430 ##STR00004## X:E:B30 H COOH H 135 0.39
370 ##STR00005## X:E:B30 dimethylester H CO.sub.2CH.sub.3 H Inact*
2.5 398 ##STR00006## Phenol Series X:E:B34 dimethylester H
CO.sub.2CH.sub.3 OH 119 3.4 430 ##STR00007## X:E:B04 H H OH 3100*
2.0 314 ##STR00008## 2'-HO-X:E:B04 OH H OH 9 Nd 330 ##STR00009##
2-CH3O-X:E:B04 OCH.sub.3 H OH 27 2.6 344 ##STR00010## X:E:B04
bis(4-methoxy) H H OCH.sub.3 Inact* 2.3 342 ##STR00011##
2'-HO-X:E:B04bis(4-methoxy) OH H OCH.sub.3 Inact nd 358
##STR00012## 2'-CH3O-X:E:B04 bis(4-methoxy) OCH.sub.3 H OCH.sub.3
Inact* Nd 372 ##STR00013## *= compound only partially soluble at
this concentration "Inact" = no significant inhibition of [.sup.3H]
CG binding to A.beta.(1-40) at 10 .mu.m "nd" = logP.sub.oct value
not determined
[0043] Some of these CG derivatives (e.g., Methoxy-X04 (Formula X)
and X34 (Formula II)) exhibit native fluorescence, which, when
combined with the compounds' high binding efficiency for A.beta.
aggregations, makes them suitable for use as a fluorescent contrast
agents for detection of A.beta. aggregations in tissue. Upon
binding to A.beta. aggregations, these CG derivatives alter the
size and mass of the aggregations. Because quasi-elastic light
scattering has a theoretical sensitivity of particle radius to the
6.sup.th power, binding of these molecules to small beta amyloid
aggregations may increase the size of these aggregations, thereby
allowing them to reach the sensitivity of detection. Thus, these
CG-derivative compounds may also have a role as size-based contrast
agents that may be detectable using light scattering techniques
such as quasi-elastic light scattering.
[0044] The Chrysamine G derivative compounds described herein have
previously been used as contrast agents for in vitro and in vivo
imaging of A.beta. aggregations present in brain tissue. When used
to image brain tissue, these derivative compounds are typically
provided in injectable form. In fact, both Klunk (Klunk et al., J
Neuropath Exp Neurology 61(9):797-805 (2002)), and Goldstein
(Moncaster et al., Alzheimer's & Dementia 2(3 Suppl. 1):S51
(2006)) have used these CG derivative imaging agents in injectable
formulations as in vivo fluorescent contrast agents. Klunk et al.
used these compounds in connection with multi-photon microscopy in
order to detect A.beta. aggregations in brain tissue of mice.
Specifically, Klunk found that doses of 10 mg/kg of Methoxy-X04
(Formula X), when administered via intravenous or intra-peritoneal
injection, provided sufficient bioavailability for use as an
A.beta.-specific contrast agent. Similarly, Goldstein injected
comparable levels of Methoxy-X04 (Formula X) into the tail of
transgenic 2576 mice and found that the compound could be detected
in the supranuclear region of the lens. However, this compound
could only be detected using extraordinary ex vivo methods, namely
2-photon fluorescent microscopy at light levels that caused
destruction of tissue samples.
[0045] Moreover, in both cases, in order to achieve sufficient
solubility for systemic injections, the investigators used dimethyl
sulfoxide (DMSO) to dissolve Methoxy-X04. However, those skilled in
the art will appreciate that DMSO is not a
pharmaceutically-acceptable solvent for use in pharmaceutical
compositions because of its action as a "carrier" chemical that is
able to carry potentially harmful chemicals into the body.
Moreover, because of the nature of DMSO solubility, it is difficult
to reconstitute dissolved compounds out of DMSO solution.
[0046] Likewise, systemic injection of CG derivative compounds for
detection of A.beta. aggregates in the supranuclear and deep
cortical regions of the eye has several critical limitations, e.g.,
1) the required doses of contrast agent are very close to the
published LD.sub.50 for these compounds, which raises the risk of
significant systemic toxicity; 2) IV injection of these compounds
results in broad systemic distribution and retention, thereby
reducing the local bioavailability for a specific target tissue
(i.e., the eye); 3) the bioavailability of a systemically
introduced contrast agent will be further degraded because there is
poor perfusion of the lens of the eye; and 4) the poor solubility
parameters of these compounds limits the dosages that can be
administered systemically without the use of unacceptable solvents,
such as DMSO.
[0047] To address these limitations, the invention provides
ophthalmic formulations that are more bioavailable to lens tissues,
have reduced systemic toxicity, and do not contain solvents that
are not suitable for clinical use.
Ophthalmic Formulations
[0048] In order to maximize lens bioavailability, such an
ophthalmic formulation exploits the filtering nature of ophthalmic
delivery. The eye is a composite structure consisting of
alternating hydrophilic (i.e. tear duct and aqueous humor) and
hydrophobic (i.e. cornea and lens) layers. Specifically, the
cornea, which transmits and focuses light into the eye, is mainly
comprised of collagen and lipid molecules. Behind the cornea is the
aqueous humor, which is a thin, watery fluid that fills the
anterior and posterior chamber of the eye and provides nutrients to
the lens and cornea epithelium. The aqueous humor is predominantly
comprised of water (>90%), while the lens of the eye is similar
to the cornea in structure (e.g., it is mainly comprised of lipid
molecules).
[0049] Thus, to successfully traverse the solubility transitions
found within the cornea, aqueous humor and lens of the eye, the
ophthalmic formulations of the invention contains solvents,
excipients, and/or carriers that help to balance the intrinsic
lipophilicity of the Congo Red or Chrysamine G derivatives. Use of
appropriate solvent(s) excipient(s), and/or carrier(s) mediates
transit through diverse microenvironments of the eye, thereby
permitting these contrast agents to reach the lens, where they will
be available to bind to A.beta. peptide aggregations present in the
supranuclear and/or deep cortical regions.
[0050] The CR- or CG-derivative compounds described herein (also
referred to herein as "active compounds"), are incorporated into
ophthalmic formulations that are suitable for administration to the
eye. Such formulations typically comprise the active compound and
one or more pharmaceutically-acceptable carriers, excipients and/or
solvents. As used herein, "pharmaceutically-acceptable carrier" or
"pharmaceutically-acceptable excipient" or
"pharmaceutically-acceptable solvent" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, that are compatible with pharmaceutical administration.
Suitable carriers are described in the most recent edition of
Remington's Pharmaceutical Sciences, a standard reference text in
the field, which is incorporated herein by reference. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compounds, use thereof in the
ophthalmic formulations described herein is contemplated.
Supplementary active compounds can also be incorporated into the
formulations, as needed.
[0051] The ophthalmic formulations of the invention are formulated
to be compatible with the intended route of administration (i.e.,
ocular administration). Solutions or suspensions used for ocular
application can include any of the following components: a sterile
diluent such as water, saline solution, fixed oils, petrolatum,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic acid
(EDTA); buffers such as acetates, citrates or phosphates, and/or
agents for the adjustment of tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide. Typically, the pH of the
formulation will be between 6 and 8. A pH of 7.4 is the most
preferred as the native pH of the tear film for ophthalmic
formulations.
[0052] In all cases, the formulations are sterile, and they are
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
viscosity fluidity is maintained, for example, by the use of a
thickening agent such as hydroxypropyl methyl cellulose. Prevention
of the action of microorganisms is achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
[0053] For ocular administration, the active compounds are
formulated into ointments (e.g., tapes), salves, gels, aqueous
solutions, eye drops, or creams, using procedures and methods
generally known in the art. To maximize transit of these ophthalmic
formulations to the lens, a pupilary dilating agent can also be
used. For example, dilation of the pupil is achieved using a
mydriatic. Examples of suitable mydriatics include, but are not
limited to atropine, cocaine, tropicamide, cyclopentolate,
homatropine, tropicamide, oxyphenonium bromide, lachesine chloride,
scopolamine (for short duration dilation), or any other appropriate
drug. The use of a pupil dilating agent helps to improve optical
access with the convenience of a single administration of the
ophthalmic formulation of the invention containing both the desired
contrast agent and mydriatic.
[0054] Any of the ophthalmic formulations described herein can be
included in a container, pack, or dispenser together with
instructions for administration.
[0055] Dosages
[0056] In any of the ophthalmic formulations described herein, the
CR or CG derivative compound will be present in the formulation in
a concentration less than 2%. For example, the formulation may
contain less than 1%, less than 0.5%, less than 0.25%, less than
0.10%, or less than 0.05% of the derivative compound. One preferred
formulation will contain less than 0.1% of the active compound
(e.g., 1 mg per gm of carrier). Those skilled in the art will
recognize the use of a formulation having less than 0.1% of the CR
or CG derivative is preferred for a variety of reasons including,
but not limited to, regulatory approval, maintenance of the
compound in solution, and/or cost.
[0057] When a CG derivative compound (e.g., MeX04) is formulated
for use as an in vivo contrast agent to detect A.beta. plaques
within the brain, typical doses administered may be either 10 mg/gm
or 10 mg/kg. (See Klunk et al., J. Neuropathol Exp. Neurol
61:797-805 (2002)). For ophthalmic uses, such as those contemplated
herein, the total dose of the derivative compound to be
administered is unlikely to exceed 1 mg per administration due to
solubility and utility limitations. Because the formulations are
applied topically, there is greater bioavailability in the region
of interest. Moreover, due to the limited solubility of the
compounds, topical application is largely independent of subject
body weight as there is little risk of system affect at the doses
used. Thus, for an average 70 kg patient, the total ophthalmic
dosage administered would be approximately a 14 mg/kg systemic
dose, which is nearly an order of magnitude less than the dosages
administered in the prior art.
[0058] Those skilled in the art will recognize that each Congo Red
or Chrysamine G derivative described herein has its own unique
solubility characteristics. Thus, the choice of the appropriate
solvent, excipient, and/or carrier will depend upon the
characteristics of the particular CR or CG derivative to be used in
the manufacture of a given ophthalmic formulation. Two illustrative
examples are presented herein: (i) the compound of Formula X known
as Methoxy-X04 (also referred to herein as "MeX04"), which is a
hydrophobic example and (ii) the compound of Formula II, known as
X34, which is a hydrophilic example. (See Table 1, supra). However,
other members of this class of compounds may also be considered and
employed in the ophthalmic formulations described herein. In
addition, each CR or CG derivative will likely require compound
specific carriers and/or excipients in order to prepare an
ophthalmic formulations having improved performance characteristics
for use as in vivo contrast agents. Determination of the
appropriate carriers, solvent and/or excipients for a given CR or
CG derivative is within the routine level of skill in the relevant
art.
[0059] Methoxy-X04
[0060] Methoxy-X04 (Formula X) is a fourth generation ligand
molecule derived from Congo Red. It is a compound exhibiting low
toxicity (Oral rat LD.sub.50 of .about.15 g/kg). At room
temperature, MeX04 is a yellow powder, which fluoresces upon
exposure to UV light. The logP.sub.oct for MeX04 is 2.6. While
Methoxy-X04 is insoluble in water, it does exhibit reasonable lipid
solubility, which may assist its diffusion across the cornea to the
lens of the eye. The degree of solubility for Methoxy-X04 is
characterized by the compound's K.sub.ow (or octanol water
partition coefficient), which is used to characterize the relative
hydrophilic/hydrophobic solubility of compounds whose solubility is
not affected by solvent pH or ionic characteristic. Ophthalmic
formulations containing Methoxy-X04 having a K.sub.ow of between
100 and 300 (preferably greater than 125; more preferably greater
than 200) is preferred to traverse the solubility barriers in the
eye.
[0061] The chemical structure of Methoxy-X04 is shown below
(Formula X).
##STR00014##
[0062] Methoxy-X04 selectively binds to A.beta. plaques in both in
vivo animal models as well as ex vivo human tissue. This A.beta.
binding ability, combined with the compound's native fluorescence
makes MeX04 a useful in vivo marker for A.beta. protein
aggregations found in the lens tissue of Alzheimer's patients.
[0063] Because these aggregations have been shown to accumulate
within the supranuclear and/or deep cortical regions of the eye
lens, administration to the surface of the eye is a convenient
route of administration for ophthalmic formulations containing this
compound. However, because Methoxy-X04 is insoluble in water, an
excipient consisting primarily of petrolatum and mineral oil, both
United States Pharmacopeia-National Formulary ("USP-NF") compounds,
is a suitable ophthalmic formulation.
[0064] Specifically, a mixture of approximately 85% petrolatum and
15% mineral oil is a suitable carrier. At concentrations of 0.1%
Methoxy-X04 (e.g., 1 mg active compound per gm of carrier), which
is an exemplary dose for the ophthalmic formulations, Methoxy-X04
remains in solution. The petrolatum in this ophthalmic formulation
can act as a carrier for a combination of dissolved and suspended
Methoxy-X04, by altering the aqueous portions of the eye to accept
the compound more readily by adding lipophilic material to the
solution of the eye's environment and by altering the interactions
of the eye with the compound by shielding it from the aqueous
environment. At concentrations of 10 mg/gm of carrier, Methoxy-X04
remains primarily in suspension.
[0065] The use of MeX04 at either concentration level (e.g., 1
mg/gm carrier or 10 mg/gm carrier), does not require the use of a
preservative because of the anti-microbial nature of petrolatum and
mineral oil. Nevertheless, various preservatives, including, but
not limited to, propylparaben or benzalkonium chloride, are
optionally added as additional preservative to ophthalmic
formulations containing MeX04. If added, these preservatives are
typically included in concentrations of less than about 1%.
[0066] Another method of delivery for hydrophobic compounds such as
Methoxy-X04 is to suspended the compound in a vehicle excipient
containing a suitable ophthalmic emulsifier, such as, for example,
hydroxypropyl methyl cellulose. This use of an emulsifier serves to
shield the hydrophobic compound from aqueous environments, thereby
maintaining it in a suspension/solution that is capable of
traversing diverse environments in the eye.
[0067] By way of nonlimiting example, one such aqueous solution
formulation contains 1% or less of the compound of Formula I;
surfactant such as polysorbate 80; a preservative such as
benzalkonium chloride; a tonicity agent such as sodium chloride; a
buffer such as boric acid or a salt thereof; a chelating agent such
as edentate disodium; and a viscosity agent such as hydroxypropyl
methylcellulose. When the ophthalmic formulation comprises
.about.0.1% or less of the hydrophobic compound (e.g., Methoxy-X04)
as well as .about.0.3% hydroxypropyl methylcellulose, .about.0.1%
polysorbate 80, ISSsolution (as a preservative), sterile water,
.about.0.4% NaCl, .about.1% boric acid, .about.0.2% sodium borate
10-hydrate, .about.0.03% edetate disodium dihydrate, sodium
hydroxide (NaOH) and hydrochloric acid (HCl) are acceptable
carriers.
[0068] As indicated, at concentrations of 0.1% Methoxy-X04 (e.g.,
1-mg active per gm carrier), Methoxy-X04 remains in solution.
Hydroxypropyl methylcellulose can act in part as a carrier of
microparticles in suspension for a mixture of dissolved and
suspended Methoxy-X04. This effect occurs by altering the aqueous
portions of the eye to enable them to accept the hydrophobic
compounds more readily, by adding lipophilic material to the eye's
environment and/or by altering the interactions of the compound
with the eye by shielding the hydrophobic compound from the aqueous
environment.
[0069] When employed in an aqueous formulation, various
preservatives, such as, for example, propylparaben or benzalkonium
chloride are added. Typically, such preservatives are included in
concentrations of less than 1%.
[0070] To minimize injury/irritation to the cornea during topical
application, particle size in formulation is preferably less than
25 microns in diameter, preferably less than 12 microns, more
preferably less than 6 microns in an aqueous solution. If
Methoxy-X04 is formulated into a parenteral (injectable)
formulation suitable for administration to the eye, typical
particle size distribution for safe administration is a fineness of
at least 99% less than 10 microns and at least 75% less than 5
microns. To achieve an acceptable particle size, a grinding process
is used to insure proper particle size while maximizing yield
(i.e., minimizing loss) of material following the grinding process.
Those skilled in the art will recognize that any other suitable
methods for controlling the particle size known in the art can also
be employed. By way of non-limiting example, such methods may
include crushing, milling, screening, and/or controlled growth of
crystals.
[0071] For example, Polysorbate 80 is weighed into a polypropylene
bottle, and an initial amount of sterile water for injection, USP,
is weighed into the bottle. Next, Methoxy-X04 is weighed and added
to the bottle. The current batch weight is determined, and the
final amount of sterile water for injection to adjust the
formulation to a final batch weight is added. Then, the YTZ
grinding media is added to the bottle, and the bottle is placed on
a roller mill for a minimum of 12 hours at a setting of 50%.
[0072] After the milling process, a polishing filter and Masterflex
tubing are connected to the bottom of a Millipore housing assembly,
while a high-pressure hose is attached to the top of the Millipore
housing. Using a nitrogen pressure gauge, the solution is pushed
thorough the housing and the polish filter. For example, the
pressure used is between 3 psi and 5 psi. This grinding process
yields micronized Methoxy-X04 having a mean particle size of 1.157
.mu.m with 100% of particles smaller than 10 .mu.m and greater than
75% of particles smaller than 5 .mu.m. Thus, those skilled in the
art will recognize that this exemplary milling process produces
Methoxy-X04 particles that are suitable for either topical or
parenteral formulation, without requiring the use of solvents, such
as dimethyl sulfoxide, that are incompatible with use in drug
formulations.
[0073] X34
[0074] X34 (Formula II) is a ligand molecule that is derived from
Congo Red by replacing the naphthalene sulfonic acids with
salicylic acids and the azo linkage with an ethenyl link. X34 is a
compound that exhibits low toxicity (Oral rat LD.sub.50 of
.about.15 g/kg). At room temperature, X34 is a yellow powder, which
fluoresces upon exposure to UV light. The logP.sub.oct for X34 is
0.42. Moreover, X34 is moderately soluble in water, when buffered
to the proper pH. At concentrations of 0.1% of X34 (e.g., 1 mg
active per gm of carrier), which is an exemplary clinical dose, X34
appears to remain in solution. Preferably, ophthalmic formulations
containing X34 (or other hydrophilic compounds of Formula I) have
LogD values of between 1 and 3.
[0075] The chemical structure of X34 is provided below.
##STR00015##
[0076] X34 selectively binds to .beta.-amyloid plaques in both in
vivo animal models as well as ex vivo human tissue. This fact,
combined with the compound's native fluorescence, makes X34 would a
useful in vivo marker for .beta.-amyloid aggregations found in the
lens tissue of Alzheimer's patients. Because these protein
aggregations accumulate in the deep cortical and/or supranuclear
regions of the lens of the eye, the eye would be the most
convenient route of administration for ophthalmic formulations
containing this compound.
[0077] A buffered, aqueous excipient is used in the preparation of
a suitable ophthalmic formulation containing X34 as the contrast
agent. The presence of the buffer provides proper pH for maximum
solubility of the compound of Formula I, and the formulation may
also contain a chelating agent to improve stability as well as a
preservative. Specifically, the buffer is, for example, Tris, the
chelating agent is, for example, ethylenediamine-tetraacetate, and
the preservative is, for example, parabens. For example, one
preferred aqueous solution formulation described herein may contain
1% or less of the compound of Formula I; a solvent such as water;
0.001% to 10% (e.g., 0.001%, 0.005%, 0.010%, 0.05%, 0.1%, 0.5%, 1%,
2.5%, 5%, 7.5%, or 10%) Tris-buffer; 0.001% to 1% (e.g., 0.001%,
0.005%, 0.010%, 0.025%, 0.05%, 0.1%, 0.5%, 0.75%, or 1%) EDTA; and
0.0001% to 1% (e.g., 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%,
0.05%, 0.1%, 0.11%, 0.5%, or 1%) parabens. In another example, a
mixture of approximately 0.1% X34 (as an active ingredient), water
(as a solvent), 2% propylene glycol (as a co-solvent for
preservative), 0.5% Tris(hydroxymethyl)aminomethane (Tris-buffer)
(as a buffer to provide proper pH for maximum solubility of X34),
0.025% ethylenediamine-tetraacetate dihydrate (EDTA Dihydrate) (as
a chelating agent to improve stability of formulation), and a total
of approximately 0.11% mixed Parabens (as a preservative) are
useful excipients.
[0078] Various other preservatives, including, but not limited to,
for example, benzalkonium chloride or parabens in the X34
ophthalmic formulations described herein. If added, these
preservatives are used in the ophthalmic formulations in
concentrations of less than 1%.
[0079] The use of a thickening agents, including, but not limited
to cellulose derivative thickening agents such as hydroxypropyl
methylcellulose, methylcellulose, hydroxyethyl cellulose and
non-cellulose agents such as polyvinylpyrrolidone, polyacrylates,
and carbomes in these X34-containing aqueous ophthalmic
formulations improve the ease of administration and/or to improve
residence time of the contrast agent within the eye. Using these
thickening agents, the viscosity of ophthalmic formulations
containing X34 is increased to not more than 1,000,000 centiPoise.
Optimally, the viscosity can be increased to approximately 10-1000
centipoise.
[0080] Additional Ophthalmic Delivery Vehicles
[0081] Other ophthalmic delivery vehicles, such as liposome
encapsulated Congo Red or Chrysamine G derivative,
micro-encapsulations or other vehicles may be employed to improve
transport of Congo Red or Chrysamine G derivatives and the
ophthalmic formulations described herein from the cornea through
the aqueous humor to the lens of the eye. Electrophoresis,
ultrasound phoresis, or other phoretic techniques are optionally
used to improve transport to a target anatomical location in the
eye.
[0082] In some embodiments, the ophthalmic formulations are
compounded with carriers that protect the compound against rapid
elimination from the body, such as controlled release formulations,
including implants, microencapsulated delivery systems, and the
like. Biodegradable, biocompatible polymers can also be used, such
as, for example, ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Methods for preparation of such formulations are known to those
skilled in the art. The materials are commercially available from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions are useful as pharmaceutically acceptable carriers and
can be prepared according to methods known to those skilled in the
art, for example, as described in U.S. Pat. No. 4,522,811.
Ocular Detection of A.beta. Protein Aggregates
[0083] The ophthalmic formulations described herein are used in
conjunction with any optical imaging or detection devices, which
collect data from the lens. (See, e.g., U.S. Pat. No. 7,107,092,
herein incorporated by reference in its entirety). Aggregates are
detected non-invasively, i.e., using a device or apparatus that is
not required to physically contact ocular tissue.
[0084] The invention includes methods of diagnosing an
amyloidogenic disorder or a predisposition thereto in a mammal, by
illuminating mammalian lens tissue with an excitation light beam
and detecting scattered or other light signals emitted from the
tissue that are known in the art. Aggregates are detected with
quasi-elastic light scattering techniques (a.k.a. dynamic light
scattering), Raman spectroscopy, fluorimetry, and/or other methods
of analyzing light returned from the test tissue. An increase of
scattered light emitted from the cortical and/or supranuclear
regions of an ocular lens indicates that the mammal is suffering
from, or is at risk of developing an amyloidogenic disorder such as
AD. Excitation light is in the range of 350-850 nm. Preferably, the
excitation light beam is a low wattage laser light such as one with
a wavelength of 450-550 nm. Those skilled in the art will recognize
that it is desirable to choose an excitation light wavelength that
avoids lens autofluorescence, which typically emits at wavelengths
of about 450 nm. For example, the excitation light beam, which
would produce an emission wavelength of approximately 500.+-.20 nm
is preferred. Alternatively, the excitation light beam is in the
very near-UV (392-400 nm) or visible (400-700 nm) ranges.
[0085] Detection of protein aggregation or accumulation or
deposition of amyloidogenic proteins or peptides in the
supranuclear/cortical region of an ocular lens is ratiometrically,
volumetrically, or otherwise mathematically compared to the same or
similar measurements in the nuclear or other regions of the lens.
These methods are useful to measure protein aggregation or
accumulation or deposition of amyloidogenic proteins or peptides in
other ocular tissues, including but not limited to the cornea, the
aqueous humor, the vitreous humor, the lens, e.g., the supranuclear
or deep cortical region of the lens, and the retina.
[0086] The QLS technique is used to non-invasively detect and
quantitate lens protein aggregation in this animal model of AD and
in human subjects. An additional advantage to using this technique
is the ability to monitor disease progression as well as
responsiveness to therapeutic intervention. A.beta.-associated lens
aggregates are found exclusively in the cytoplasmic intracellular
compartment of human lens cells, specifically lens cortical fiber
cells in contrast to A.beta. deposits in the brain, which are
largely extracellular. A.beta. fosters human lens protein to
aggregate through metalloprotein redox reactions and this
aggregation by chelation or antioxidant scavengers.
[0087] The major proteins that can scatter light in a human eye
lens are .alpha.-, .beta.-, .gamma.-crystallins. Since the
crystallins are abundant and large molecules (molecular weight
.about.10.sup.6 Daltons), they induce the greatest amount of
scattering of light, including laser radiation in dynamic light
scattering (DLS) measurements. When the lens protein molecules are
aggregated, they give rise to lens opacities. The lens gradually
becomes cloudy as a result of light scattering and absorbance,
thereby hindering light transmission and the ability to focus a
sharp image on the retina at the back of the eye.
[0088] Methods for measuring DLS, are known in the art, e.g.,
Benedek, G. B., 1997, Invest. Opthalmol. Vis. Sci. 38:1911 1921;
Betelhiem, et al., 1999, J. Biochem. Biophys. Res. Comm. 261(2):292
297, Ansari et al., Diabetes Technol. Ther. Summer 1(2): 159-68
(1999); and U.S. Pat. No. 5,540,226. For example, a monochromatic,
coherent, low-powered laser is shined into the lens of a subject
such as a human patient. Agglomerated particle dispersions within
the lens reflect and scatter the light. Light scattering is
detected using a variety of known methods such as photo multiplier
tube, a solid-state photo diode or a charge coupling device.
Because of random, Brownian motion of the lenticular protein
crystallins, the concentration of the crystallins appears to
fluctuate, and hence, the intensity of the detected light also
fluctuates. However, a temporal autocorrelation function of the
photo current is mathematically analyzed to reveal the particle
diffusivity. The data reveals the composition and extent of
cataractogenesis. An increase in light scattering in the
supranuclear and/or cortical region of the lens (alone and/or
normalized to the scattering in the lens nucleus, where general
aging effects on the lens predominate and/or normalized for age)
compared to a known normal value or a normal control subject
indicates the presence of protein aggregation associated with a
neurodegenerative disease such as AD. This finding, in turn, serves
as a biomarker for the AD disease process and hence is of clinical
utility in the diagnosis, prognosis, staging, and monitoring of the
AD or other amyloidogenic disorders.
EQUIVALENTS
[0089] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
* * * * *